1. Field of the Invention
The present invention relates to a polypeptide which specifically binds to the .gamma.-chain of human interleukin-2 receptor to selectively inhibit the binding of the .gamma.-chain of human interleukin-2 receptor to the .beta.-chain of the same. The polypeptide has the biological activity of blocking the human interleukin-2 response. The invention also relates to an immunosuppressant containing the polypeptide, a DNA coding for the polypeptide, a recombinant DNA having the gene, a transformant having the recombinant DNA, and a method for producing the intended polypeptide by cultivating the transformant.
The polypeptide of the present invention is a valuable substance which is usable, independently or along with substances capable of inhibiting the binding of interleukin-2 to interleukin-2 receptor, as a medicine effective in preventing the rejection after transplantation and also in curing inflammatory diseases such as allergic diseases and autoimmune diseases, the possibility that interleukin-2 will participate in the rejection and also in such inflammatory diseases having been suggested. "Interleukin-2" may be hereinafter referred to as "IL-2".
2. Discussion of the Background
Now that surgical techniques for transplantation have been improved noticeably, success in transplantation essentially depends upon inhibiting the rejection of a graft after the operation. The rejection phenomenon results from a series of immunoreactions occurring through the interaction of the graft with organs of the recipient which have recognized the graft as foreign matter and reject them. Therefore, so-called immunosuppressants such as various steroids, azathiopurine, methotrexate and 6-mercaptopurine have heretofore been used as rejection-inhibiting medicines. However, since the safety range of such medicines is narrow and their effects are weak, extreme improvement in the take of grafts in recipients could not be attained.
Using cyclosporin A, a drug which has been developed recently, the take of grafts in recipients has improved surprisingly. However, it has been recognized that cyclosporin A has serious nephrotoxicity, which has obliged us to limit its use. Given this situation, it is desirable to develop more safe, more specific and more effective immunosuppressants.
IL-2 is a protein that is produced by helper T-cells and is an extremely important factor for the host defense machinery, having various functions of broad range. For example, it is involved in induction of proliferation and differention of killer T-cells and induction of differention of B cells in living bodies. It has been said that killer T-cells activated by IL-2, etc. participate strongly in the host vs. graft reaction (HVG reaction) or the graft vs. host reaction (GVH reaction) which is considered to be the key to the take of grafts in transplantation of organs or bone marrow.
On the other hand, it is considered that autoimmune diseases are caused by an imbalance of the immune system in the living body, resulting in the body attacking itself. In particular, there is a great possibility that excess production of factors participating in the immune system, such as IL-2, as well as excess reaction thereto, will essentially cause an imbalance of the immune system.
Thus, it is possible that if the IL-2 response may be selectively and effectively inhibited, then rejection after transplantation may be prevented and autoimmune diseases may be cured. In fact, there have been published reports in which IL-2 and a cytotoxin were fused together to prepare a polypeptide capable of selectively damaging IL-2 responding cells having IL-2 receptors. The polypeptide was administered to a rat with adjuvant arthritis, which is one of the animal models with an autoimmune disease, with the result that the onset of the symptoms of arthritis was delayed in the rat and the presented symptoms of arthritis were slight. The report further revealed that when the polypeptide was administered, during the transplantation, to a mouse that had received a cardiac graft by transplantation from an allogenic mouse, then rejection of the transplanted cardiac graft in the recipient mouse was inhibited. (Proc. Natl. Acad. Sci. USA, Vol. 86, page 1008, 1989.)
However, the polypeptide obtained by fusing IL-2 and a cytotoxin has a short half-life period in blood so that a large amount of the polypeptide must be dosed in order to attain the intended effect, which, however, also causes some harmful side effects. Therefore, the development of a medicine which is safer, more specific and more effective in inhibiting the IL-2 response has been desired.
Heretofore, it has been known that the IL-2 receptor on IL-2 responding cells is composed of two glycoprotein molecules, one being an .alpha.-chain having a molecular weight of about 55 kd and the other being a .beta.-chain having a molecular weight of about 75 kd. The dissociation constant of binding between these molecules and IL-2 is 10.sup.-8 M for the .alpha.-chain and 10.sup.-9 M for the .beta.-chain, respectively. It has been considered that, when both the .alpha.-chain and the .beta.-chain are bound to IL-2 at the same time, then the resulting association has high-affinity binding with a dissociation constant of 10.sup.-12 M.
However, it has recently been clarified that, even when a human .beta.-chain cDNA is transfected into mouse non-lymphocytic cells, it does not bind to IL-2, and that even when both human .alpha.-chain and .beta.-chain cDNA's are transfected into the same, they may bind to IL-2 only to an intermediate degree without forming any high-affinity binding to the same. (Science, Vol. 244, page 551, 1989.) Hence, the existence of a third molecule different than the .alpha.-chain and the .beta.-chain, which is crucial for the binding to IL-2, has been suggested.
We, the present inventors have cloned a gene coding for a glycoprotein molecule having a molecular weight of 64 kd which is the third molecule constituting the IL-2 receptor complex (hereinafter referred to as IL-2 receptor .gamma.-chain) (Japanese Patent Application No. 4-104947 and Science, Vol. 257, page 379, 1992) by which the IL-2/IL-2 receptor system has been clarified completely.
Specifically, it has been shown that the association formed by transfection of both human .beta.-chain and .gamma.-chain cDNA's into mouse non-lymphocytic cells shows an intermediate degree of binding between them, which was previously attributed to the .beta.-chain only; and that the transfection of all human .alpha.-chain, .beta.-chain and .gamma.-chain cDNA's into the same cell gives high-affinity binding between them. The latter effect has previously been attributed to the .alpha.-chain and the .beta.-chain only. In addition, it has been shown that the internalization of an IL-2/IL-2 receptor complex into cells, which is considered to be a prerequisite for signal transduction for IL-2, does not occur in cells expressing only the .beta.-chain or in cells expressing both the .alpha.-chain and the .beta.-chain, but may occur in cells additionally expressing the .gamma.-chain. Accordingly, the present inventors have clarified for the first time that the IL-2 receptor .gamma.-chain molecule is not a molecule which merely defines its binding to IL-2, but is a molecule which is indispensable for the signal transduction triggered by IL-2.
It is known that the IL-2 receptor .gamma.-chain molecule binds to the extracellular site in the IL-2 receptor .beta.-chain in the presence of IL-2. (Science, Vol. 257, page 379, 1992.) Therefore, it is considered that if the binding between the .beta.-chain and the .gamma.-chain were inhibited, the .beta.-chain alone could not bind to IL-2, or both the .alpha.-chain and the .beta.-chain might bind to IL-2 to some intermediate degree while the IL-2-signal transduction would be blocked. Namely, if the binding between the IL-2 receptor .beta.-chain and the IL-2 receptor .gamma.-chain on IL-2 responding cells is inhibited, the IL-2 signal transduction can be completely blocked and the cells then become unresponsive to IL-2. Therefore, by the inhibition, the rejection of transplanted tissue, in which IL-2 is believed to participate, can be prevented and autoimmune diseases, in which IL-2 is considered to also actively participate, can be cured. Up to the present time however, no substance having the activity of inhibiting the binding between the IL-2 receptor .beta.-chain and the IL-2 receptor .gamma.-chain was known.